A critical area of research for the development of new interventions to halt HIV transmission is the characterization of the immunologic environment of the female reproductive tract (FRT). Globally, greater than 90% of HIV is transmitted following heterosexual intercourse and women are twice as likely to contract HIV from heterosexual sex than men. Mucosal tissues in the FRT therefore represent the frontline of transmission since they are the anatomic site at which HIV infection is first established in most new transmission cases. Despite this, HIV has largely been studied in the peripheral blood, which contains just 2% of lymphocytes and has been shown to have distinct immunologic features relative to those in the FRT. Based upon studies in nonhuman primates, soon after HIV exposure viral replication occurs within the mucosa of the cervix and vagina for 5-7 days prior to systemic dissemination. This early period in HIV transmission has been referred to as a window of opportunity, since interventions which target HIV early in the FRT could prevent systemic viral dissemination. These observations suggest that the development of effective strategies to prevent HIV in women will require a detailed understanding of the critical FRT mucosal factors influencing HIV susceptibility. Recent results from the CAPRISA 004 vaginal microbicide trial suggest that elevated FRT inflammation increases risk of HIV acquisition by up to 14-fold. Microbially-driven sexually transmitted infections (STIs), bacterial vaginosis (BV), and reproductive hormones are known modulators of genital inflammation and increased HIV acquisition risk, suggesting the vaginal microbiome more broadly as well as reproductive hormones may play important roles in engendering FRT inflammation. The characterization of the vaginal microbiome to date has largely focused on the bacterial component, however other domains of life, including fungi, viruses, and potentially as-yet-incompletely-defined organisms, inhabit the FRT and may contribute to inflammation. New high throughput sequencing (HTS) technologies can comprehensively characterize the microbiome but have not been applied to the assessment of the vaginal microbiome beyond assessment of bacteria. The mechanism by which reproductive hormones, including endogenous progesterone and depot medroxyprogesterone acetate (the most common form of hormonal contraception used in sub-Saharan Africa), increase HIV acquisition remains incompletely understood. Collectively, these data indicate that both microbial and hormonal factors can significantly affect FRT inflammation and therefore potentially increase HIV acquisition in women. To fully define optimal strategies to prevent HIV transmission, a more complete understanding of the interdependent role of the vaginal microbiome, reproductive hormones, and genital inflammation is needed.